FIG. 4.

18 E6* is expressed and associates in vivo with Flag-PATJ. (A) Alignment of FL 18 E6 and 18 E6* ORFs. Splicing of 18 E6 removes an intron between nucleotides (nt) 132 and 310 and results in an alternative 3′ reading frame. The zig-zag lines represent DNA from the Myc-tag vector. The arrows identify the binding sites for the 5′ and 3′ primers used for RT-PCR. (B) RT-PCR was performed on total RNA collected from approximately 1.5 × 10⁶ 293T cells transiently transfected with 1 μg of pMycL.1-18 E6 WT or 18 E6 SD construct. PCR was also performed using pMycL.1-18 E6 WT vector DNA and the same primers. The PCR products were resolved on an agarose gel and detected by ethidium bromide staining. The sizes of the DNA molecular weight markers (M) are indicated. Approximately 2 × 10⁶ 293T cells were transiently transfected with 2 μg of Flag-PATJ expression plasmid and 3 μg of pMycL.1 plasmid or pMycL.1 constructs containing 18 E6 WT, SD, or E6*. (C and D) Equal amounts of protein, extracted after 48 h, were either resolved directly (C) or bound to Flag affinity beads (D). The complexes bound to Flag affinity beads were washed extensively and eluted. Following SDS-PAGE, the whole-cell lysates and Flag-bound complexes were probed with antibody to Flag, Myc, and actin. Arrows identify Flag-PATJ, Myc-E6 FL, Myc-E6*, and actin.